This invention relates to the control of the sex of the offspring of mammals by the separation of male-(Y) and female-(X) determining sperm.
Any separation technique, to be effective, must be dependent on some phenotypic difference between X- and Y-sperm Suggested distinctions include fluorescence of the Y-chromosome, weight, density, size, adherence to Sephadex, motility in albumin, pH, surface charge, cell surface antigens, and enzymatic and hormonal environment. Erickson and Glass, Functional Differences Between Sperm Bearing the X- or Y- Chromosome, in PROSPECTS FOR SEXING MAMMALIAN SPERM (1982). For example, Kaneko, et al., Biochem. & Biophys. Res. Commun., 124: 950 (1984) suggests that X- and Y-bearing sperm may differ in cell surface charge as a result of differences in cell surface sialic acid content.
Considerable skepticism exists as to whether a successful separation scheme is even possible. Gledhill, Fertility and Sterility, 40: 572 (1983). Nonetheless, many patents have been issued to workers in this field. Bovenkamp, U.S. Pat. No. 3,687,806; Ericsson, U.S. Pat. No. 4,009,260; Hancock, U.S. Pat. No. 4,085,205; Bhattacharya, U.S. Pat. No. 4,155,831; Lawson, U.S. Pat. No. 4,276,139 and U.S. Pat. No. 4,225,405; Lang, U.S. Pat. No. 4,083,957; Shrimpton, U.S. Pat. No. 3,894,529 and U.S. Pat. No. 4,327,177; Adair, U.S. Pat. No. 4,362,246.
Albertsson generally teaches that polymer phase partition chromatography may be used to separate cells. He broadly suggests that the system may be designed so that cells are separated according to electrochemical, hydrophobic or biospecific surface properties. Albertsson, Endeavor, 1: 69 (1978). Walter and Krob, Biochem. & Biophys. Res. Commun., 120: 250 (1984) detected differences in membrane surface properties of erythrocytes by partitioning in dextran-poly(ethylene glycol) aqueous phase systems subject to countercurrent distribution. An improvement in their methodology was reported in Cell Biophysics, 6: 253 (1984). See also Fisher and Walter, Biochem. & Biophys. Acta, 801: 106 (1984); Walter and Krob, Biochem. & Biophys. Acta, 855: 8 (1986). Sharpe, et al., Bioscience Repts. 4: 415 (1984) partitioned human bone cells in the same two-phase system, obtaining two cell populations with different growth rates. Sharpe and Watts, J. Cell Sci., 75: 339 (1985) used aqueous two phase partition to detect changes in the cell surface properties of D. discoideum amoebae.
Immunoaffinity partitioning was explored by Sharpe, et al., Anal. Biochem. 154: 110 (1986), who used a two-phase dextran/PEG system in conjunction with PEG-bound antibody. The antibody "dragged" the antigen into the PEG-rich phase, thus accentuating the partitioning of the antigen achieved by the two-phase system alone.
Hammerstedt, in Prospects for Sexing Mammalian Sperm 169 (1982) speculates that polymer phase partition chromatography could be applied to the separation of sperm if a differentiating property could be identified.
Separation by immunological means has been suggested. Bryant, U.S. Pat. No. 4,448,767. However, for this method to succeed, one must develop an antibody against a cell surface marker for an X- or Y-determining sperm. McCormick, Infertility, 5(3): 217-227 (1983) exposed sperm to polyclonal antibodies against the H-Y antigen, which is expressed by male sperm. Monoclonal antibodies were similarly used when they became available. However, the efficacy of this method has been questioned. Hoppe and Koo, J. Reprod. Immunol., 6: 1-9 (1984).
Antibodies have been prepared against sperm surface antigens. Smith, et al., J. Exper. Zoology, 225: 157 (1983); Myles, et al., Cell, 23: 433 91981); Peterson, et al., J. and Sterility, 37: 249 (1982); Schmell, et al., J. Reprod. Immunol., 4: 91 (1982). In particular, antibodies have been prepared against the sulfogalactoglycerolipid found in mammalian testes. Lingwood, et al., J. Immunology, 124: 769 (1980); Lingwood and Schactiter, J. Cell Biol., 89: 621 (1981); Lingwood, et al., Can. J. Biochem., 59: 556 (1981); Shirley and Schacter, Can. J. Biochem., 58: 1230 (1980). However, these antibodies have not been used in separating X- and Y-sperm.
It has also been suggested that cryptic spermal antigens may be exposed by treatment with pronase, trypsin, or other enzymes, though care must be taken to ensure that the sperm can still recognize and bind specifically to oocytes. See Nicholson, The Sperm Membrane, in PROSPECTS FOR SEXING MAMMALIAN SPERM (1982) at 45.